Nutrition Research and Practice (Nutr Res Pract) 2013;7(5):352-358 http://dx.doi.org/10.4162/nrp.2013.7.5.352 pISSN 1976-1457 eISSN 2005-6168

Impact of Korean on weight gain and immune responses in high- diet-induced obese mice

Soyoung Park1, Yeseo Lim1, Sunhye Shin1 and Sung Nim Han1,2§ 1Department of Food and Nutrition, College of Human Ecology, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea 2Research Institute of Human Ecology, Seoul National University, Seoul 151-742, Korea

Abstract Korean (PNO) has been reported to have favorable effects on metabolism and appetite control. We investigated whether PNO consumption could influence weight gain, and whether the PNO-induced effect would result in an improvement of immune function in high-fat diet (HFD)-induced obese mice. C57BL/6 mice were fed control diets with 10% energy fat from either PNO or (SBO), or HFDs with 45% energy fat from 10% PNO or SBO and 35% , 20% PNO or SBO and 25% lard, or 30% PNO or SBO and 15% lard for 12 weeks. The proliferative responses of splenocytes upon stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS), Con A-stimulated production of interleukin (IL)-2 and interferon (IFN)-γ, and LPS-stimulated production of IL-6, IL-1β, and prostaglandin E2 (PGE2) by splenocytes were determined. Consumption of HFDs containing PNO resulted in significantly less weight gain (17% less, P < 0.001), and lower weight gain was mainly due to less white adipose tissue (18% less, P = 0.001). The reduction in weight gain did not result in the overall enhancement in splenocyte proliferation. Overall, PNO consumption resulted in a higher production of IL-1β (P = 0.04). Replacement of SBO with PNO had no effect on the production of IL-2, IFN-γ, IL-6, or PGE2 in mice fed with either the control diets or HFDs. In conclusion, consumption of PNO reduced weight gain in mice fed with HFD, but this effect did not result in the overall improvement in immune responses.

Key Words: Pine nut oil, obesity, high-fat diet, immune response, inflammatory cytokine

Introduction2) by Sugano et al. [5], consumption of PNO (22% energy) alleviated high blood pressure in spontaneously hypertensive rats Pine nuts, which are oily seeds of the genus pinus, have been after five weeks of feeding. Hughes et al. [6] showed that utilized for culinary purposes around the world for centuries. providing a PNO capsule prior to an ad libitum buffet lunch Pinus koraiensis, a native plant of eastern Asia and commonly resulted in reduced food intake in overweight female subjects. called the Korean pine nut, is one of the main types of Pasman et al. [7] observed that cholecystokinin and glucagon-like commercial pine nuts. Lipid content of the Korean pine nut peptide-I secretion were higher in subjects who received a PNO comprises 62% of the total weight of the nut [1]. Pine nuts capsule than in those who received a placebo, thereby supporting contain fatty acids with an unusual structure, e.g., Δ5-unsaturated the findings by Hughes et al. [6]. polymethylene-interrupted fatty acids (Δ5-UPIFAs). These fatty Dietary fatty acids, especially polyunsaturated fatty acids acids are the characteristic components of obtained from (PUFAs), play a major role in regulating immune function by conifer seeds and leaves [2]. (18:3, Δ5,9,12) is modulating the production of lipid mediators involved in a variety a major Δ5-UPIFA present in pine nuts. The Korean pine nut of signaling pathways. In general, n-6 PUFAs are known to oil (PNO) contains 5% (16:0), 2% promote hyperactive immune responses by providing substrates (18:0), 27% (18:1, Δ9), 45% (18:2, Δ for proinflammatory lipid mediators; whereas consumption of n-3 9,12), 1% eicosenoic acid (20:1, Δ11), 15% pinolenic acid, and PUFAs results in the alleviation of proinflammatory responses 3% other Δ5-UPIFAs [2]. [8]. However, γ-linolenic acid (18:3, Δ6,9,12), which is an n-6 The health benefits of PNO have been investigated in several PUFA, was reported to suppress chronic inflammation by studies. Asset et al. [3,4] reported that PNO lowered the total increasing the cellular levels of dihomo-γ-linolenic acid (20:3, cholesterol and levels in animal models. In a study Δ8,11,14) [9]. Dihomo-γ-linolenic acid can compete with

This work was supported by a grant from the National Research Foundation (NRF) of Korea, funded by the Ministry of Education, Science and Technology (grant number 2010-0024878). § Corresponding Author: Sung Nim Han, Tel. 82-2-880-6836, Fax. 82-2-884-0305, Email. [email protected] Received: March 29, 2013, Revised: May 17, 2013, Accepted: May 20, 2013 ⓒ2013 The Korean Nutrition Society and the Korean Society of Community Nutrition This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Soyoung Park et al. 353 (20:4, Δ5,8,11,14) as a substrate for cyclooxy- Table 1. Composition of experimental diets (g)1) genase and . Control High-fat Pinolenic acid, which is the positional isomer of γ-linolenic (10% kcal Fat) (45% kcal Fat) 10% Oil 20% Oil 30% Oil acid, can also be taken up and elongated to form eicosatrienoic 10% Oil +35% Lard +25% Lard +15% Lard acid (20:3, Δ7,11,14) [10]. Chuang et al. [11] reported that Casein 200 200 200 200 pinolenic acid, taken up by cells, modified the L-Cystine 3 3 3 3 composition of cellular phospholipids, resulting in a decreased Sucrose 350 172.8 172.8 172.8 production of proinflammatory eicosanoids in the murine Cornstarch 315 72.8 72.8 72.8 macrophage cell line. In contrast to the in vitro results, the Dyetrose2) 35 100 100 100 production of proinflammatory lipid mediators was elevated in PNO3) or SBO 45 45 90 135 animals that were fed with PNO [5,12]. In a study that examined Lard 0 157.5 112.5 67.5 the effects of a five-week PNO diet (22% energy) on immune t-Butylhydroquinone 0.009 0.009 0.018 0.027 + parameters [12], the relative proportion of splenic CD4 T Cellulose 50 50 50 50 lymphocytes, the splenic production of immunoglobulin G and Mineral mix4) 35 35 35 35 5) E, and the leukotriene B4 released from peritoneal exudate cells Vitamin mix 10 10 10 10 were higher in rats that were fed with PNO compared with those Choline bitartrate 2 2 2 2 fed with oil (a source of linoleic acid; n-6, 18:2, Δ9,12) Total 1,045 848.1 848.1 848.1 or evening primrose oil (a source of γ-linolenic acid; n-6, 18:3, kcal/g diet 3.69 4.64 4.64 4.64 Δ6,9,12). Therefore, pinolenic acid has the potential to modulate 1) Resource: Dyets, Inc, Bethlehem, PA, USA. 2) the immune response via a mechanism that may be different from Dyetrose (Dyets) is dextrinized cornstarch containing 90-94% tetrasaccharides. 3) PNO was a gift from Dubio Co., Ltd. (Gyeonggi-do, Korea). that of other n-6 PUFAs in vivo. However, information regarding 4) Thirty-five grams of mineral mix (Dyets, #210099) provides 5.1 g calcium, 4 g the effect of PNO on immune function is limited. phosphorus, 3.6 g potassium, 1 g sodium, 1.6 g chloride, 0.5 g magnesium, 0.3 g sulfur, 59 mg manganese, 46 mg iron, 25 mg zinc, 5 mg copper, 0.2 mg Obesity has been associated with impaired immune function. selenium, 0.2 mg iodine and 4.2 g sucrose. It was reported that obese patients had an increased risk of 5) Ten grams of vitamin mix (Dyets, #300050) provides 4,000 IU vitamin A, 1,000 infection [13] and poor antibody response to vaccination [14]. IU vitamin D3, 50 IU vitamin E, 30 mg niacin, 16 mg pantothenic acid, 7 mg vitamin B6, 6 mg vitamin B1, 6 mg vitamin B2, 2 mg folic acid, 0.8 mg menadione, 0.2 It was suggested that altered levels of circulating hormones and mg biotin, 10 μg vitamin B12 and 9.8 g sucrose. nutrients, such as glucose and lipids, might contribute to dysregulation of the immune cells [15]. Several studies showed 10% kcal fat from PNO or soybean oil (SBO), e.g., 10% PNO that adequate weight reduction could correct the altered immune or SBO (PC, SC). The following six different HFDs contained response [16,17], which suggests that immune response could a total of 45% kcal from fat: 10% kcal from PNO or SBO + be improved by weight reduction in obese subjects. 35% kcal from lard (P10, S10), 20% kcal from PNO or SBO A study by Ferramosca et al. [18] showed that PNO consump- + 25% kcal from lard (P20, S20), and 30% kcal from PNO or tion led to less weight gain in mice that were fed with a 29% SBO + 15% kcal from lard (P30, S30). PNO was a gift from energy fat diet containing PNO (17% energy). Here, we investigated Dubio Co., Ltd. (Gyeonggi-do, Korea). The composition of whether PNO reduced weight gain in high-fat diet (HFD)-induced experimental diets is shown in Table 1. Mice were housed obese mice and whether this effect led to an improvement in individually and maintained in an animal facility with controlled the immune function. To test a dose-dependent effect, 10%, 20%, temperature (23 ± 3℃) and humidity (55 ± 10%), and a 12 h and 30% of energy was provided by PNO in HFDs (45% energy light/12 h dark cycle. SBO was chosen as the control oil because fat). The effect of PNO replacement in the control diet (10% its fatty acid composition is similar to that of PNO, except for energy fat) on immune responses was also investigated. To the its pinolenic acid content. Antioxidant was added to the diet (0.2 best of our knowledge, this is the first study that examines the μg t-butlyhydroquinone/g oil) to prevent oxidation of polyunsa- effect of PNO in both control diet and HFD on the immune turated fatty acids, and fresh diet was provided every other day. function in an animal model. The fatty acid composition of experimental diets is shown in Table 2. The body weight was recorded once a week, and food intake was measured 4 times a week. At the end of the experi- Materials and Methods mental period, mice were fasted for 12 hours and euthanized

by asphyxiation with CO2. Blood was collected via cardiac Animals and diets puncture. Serum was separated and stored at -80℃ for later analysis. Epididymal, subcutaneous and perirenal-retroperitoneal Five-week-old male C57BL/6 mice were purchased (Central fat pads were dissected and weighed. Spleens were aseptically Lab Animal Inc., Seoul, Korea) and randomly divided into 8 removed and placed in a sterile RPMI 1640 media (Lonza, groups after 3 days of acclimation. Mice were fed with experi- Walkersville, MD), supplemented with 100 kU/L penicillin mental diets for 12 weeks ad libitum. Control diets contained (Gibco, Carlsbad, CA), 100 mg/L streptomycin (Gibco), 2 mmol/L 354 Impact of Korean pine nut oil on immune function

Table 2. Fatty acid composition of experimental diets (% of fatty acids)1) SBO PNO Control High-fat Control High-fat SC S10 S20 S30 PC P10 P20 P30 (C14:0) ND 0.9 0.7 0.4 ND 0.9 0.7 0.4 Palmitic acid (C16:0) 11.9 18.9 16.4 14.0 7.0 17.8 14.5 10.5 Stearic acid (C18:0) 4.8 11.1 8.8 6.9 3.6 10.7 8.5 6.2 Total saturated fatty acid 16.7 30.9 25.9 21.3 10.6 29.4 23.7 17.1 (C16:1 Δ9) ND 1.4 1.0 0.6 ND 1.4 1.3 0.7 Oleic acid (C18:1 Δ9) 21.1 34.7 31.3 27.7 27.4 36.0 33.5 31.7 Total monounsaturated fatty acid 21.1 36.1 32.3 28.3 27.4 37.4 34.8 32.4 Linoleic acid (C18:2 Δ 9,12) 54.9 30.3 37.7 44.9 47.2 28.6 34.0 39.7 α-linolenic acid (C18:3 Δ 9,12,15) 7.4 2.8 4.2 5.5 0.8 1.3 1.1 1.0 Pinolenic acid (C18:3 Δ 5,9,12) ND ND ND ND 14.0 3.3 6.5 9.7 Total polyunsaturated fatty acid 62.3 33.1 41.9 50.4 62 33.2 41.6 50.4 ND, not detected. 1) Fatty acid composition was determined by gas chromatography method.

L-glutamine (Gibco), and 25 mmol/L HEPES (Sigma Aldrich, Cytokine and prostaglandin E2 (PGE2) production St Louis, MO) (complete RPMI). This study was approved by Splenocytes (5 × 106 cells/well) were stimulated with 5 mg/L the Animal Care and Use Committee at Seoul National University of Con A for 48 hours or 10 mg/L of LPS for 24 hours in 24-well (approval no. SNU-101029-1). plates at 37℃ in a 5% CO2 atmosphere. The cell-free superna- tants were collected and stored at -80℃. Levels of interleukin Isolation of splenocytes (IL)-2, interferon (IFN)-γ, IL-6, and IL-1β were measured by ELISA (BD OptEIA set; BD Pharmingen, San Diego, CA) Spleens were minced with the frosted ends of the microscope according to the manufacturer’s instructions. Levels of PGE slides (Fisher Scientific, Waltham, MA) in complete RPMI. After 2 were measured using a competitive ELISA kit (PGE EIA kit; centrifugation, the red blood cells were lysed using Gey’s 2 Cayman Chemicals, Ann Arbor, MI). solution, and the remaining leukocytes were washed twice with complete RPMI. The viable cells were counted by trypan blue exclusion on a hemacytometer (Fisher Scientific). Isolated Serum leptin splenocytes were resuspended in complete RPMI containing 10% Serum leptin levels were determined by ELISA (Quantikine® heat-inactivated fetal bovine serum (Gibco) for culture. ELISA kit; R&D Systems, Minneapolis, MN).

Lymphocyte proliferation Statistical analysis Lymphocyte proliferation was determined using a [3H] thymi- Statistical analysis was performed using PASW Statistics 18 dine incorporation assay. Splenocytes (4 × 105 cells/well) were (SPSS Inc., Chicago, IL). A two-way ANOVA was used to cultured in triplicate with concanavalin A (Con A) (Sigma evaluate the overall effects of the fat amount and type of fat Aldrich) at 0.5 or 1.5 mg/L (final concentration) or lipopoly- used in the diets, and the interaction between these parameters. saccharide (LPS) (Sigma Aldrich) at 5, 15, or 30 mg/L (final When the effects were significant, an LSD multiple-comparison concentration) in a 96-well flat bottom plate (Becton Dickinson, post-hoc test was performed. A Pearson correlation was used to Franklin Lakes, NJ) for 72 hours at 37℃ in the presence of 5% determine the association between the body weight, adipose CO . Each well was pulsed with 18.5 kBq of [3H] thymidine 2 tissue weight, and serum leptin level. The results from all the (Perkin Elmer, Boston, MA) for the last 4 hours of incubation. comparisons were considered significant at P < 0.05. Data were The cells were harvested on filter paper using a cell harvester reported as the mean ± SE. (FilterMate; Perkin Elmer), and radioactivity was measured using a liquid scintillation counter (MicroBeta; Perkin Elmer). The results are reported as corrected disintegrations per minute (dpm), Results which is the average dpm of the mitogen-stimulated wells minus the average dpm of wells without mitogens. Weight gain, white adipose tissue weight and energy intake Weight gain, white adipose tissue weight, and energy intake Soyoung Park et al. 355

Table 3. Body weight, weight gain, white adipose tissue weight, food intake and energy intake of mice fed with control diets or HFDs containing PNO or SBO SBO PNO Effect of Effect of Control High-fat Control High-fat fat amount, fat type, SC S10 S20 S30 PC P10 P20 P30 P value P value (n = 10) (n = 11) (n = 11) (n = 12) (n = 11) (n = 11) (n = 10) (n = 12) Body weight at 0 wk (g) 17.3 ± 0.5 17.0 ± 0.4 17.1 ± 0.3 16.8 ± 0.4 16.7 ± 0.4 17.0 ± 0.3 16.9 ± 0.4 16.7 ± 0.4 0.92 0.54 Body weight at 12 wk (g) 32.8 ± 1.0ab 38.5 ± 1.4de 39.4 ± 1.1e 38.0 ± 1.2de 30.5 ± 0.6a 34.6 ± 1.4bc 35.4 ± 1.1bcd 34.6 ± 1.2bc 0.00 0.00 Weight gain (g) 15.5 ± 0.8ab 21.5 ± 1.4cd 22.3 ± 1.0d 21.2 ± 0.9cd 13.8 ± 0.6a 17.5 ± 1.3b 18.5 ± 1.0bc 17.8 ± 1.1b 0.00 0.00 White adipose tissue 3.1 ± 0.2b 5.3 ± 0.4d 5.4 ± 0.3d 5.3 ± 0.3d 2.2 ± 0.2a 4.4 ± 0.4c 4.6 ± 0.4cd 4.2 ± 0.3c 0.00 0.00 weight (g)1) Average daily Food 3.20 ± 0.06d 2.82 ± 0.05bc 2.86 ± 0.03bc 2.89 ± 0.05c 3.20 ± 0.03d 2.76 ± 0.04ab 2.77 ± 0.03abc 2.68 ± 0.05a 0.00 0.01 intake (g/d) Average daily energy 11.8 ± 0.2a 13.1 ± 0.2cd 13.3 ± 0.1cd 13.4 ± 0.2d 11.8 ± 0.1a 12.8 ± 0.2bc 12.9 ± 0.2bcd 12.4 ± 0.2b 0.00 0.01 intake (kcal/d)2) Values are presented as the mean±SE. A two-way ANOVA was used to determine the effects of fat amount and type, followed by an LSD post-hoc test. Means in a row without a common superscript represent significant differences (P < 0.05). 1) White adipose tissue weight is the sum of the weights of epididymal, subcutaneous, and perirenal-retroperitoneal depots. 2) Average daily energy intake (kcal/d) = Average daily food intake (g/d) × Calories per g diet (kcal/g diet). were significantly affected by the amount and type of fat used in the diets. No significant interaction was observed (Table 3). Replacement of SBO with PNO resulted in less weight gain and less white adipose tissue in mice fed with HFDs. Mice in the P10 (P = 0.01), P20 (P = 0.01), and P30 (P = 0.02) groups gained less body weight than those in the S10, S20, and S30 groups, respectively, after 12 weeks on the diet. Mice in the P10 (P = 0.04), P20 (P = 0.07), and P30 (P = 0.02) groups had less white adipose tissue than those in the S10, S20, and S30 groups, respectively. PNO consumption also led to less white adipose tissue in mice fed with control diets (P = 0.05). A significantly positive correlation was observed between weight gain and white Fig. 1. Serum leptin levels of mice fed with control diets or HFDs containing adipose tissue weight (r = 0.91, P < 0.001). PNO or SBO (SC, n = 10; PC, n = 11; S10, n = 11; P10, n = 11; S20, n = 11; P20, n = 10; S30, n = 12; P30, n = 12). Values are presented as the mean±SE. Inclusion of PNO in HFD resulted in less energy intake than A two-way ANOVA was used to determine the effects of fat amount and type, the inclusion of SBO in HFD, and this was due to less food followed by an LSD post-hoc test. Labeled means without a common letter represent significant differences (P < 0.05). Serum leptin levels were determined by intake. Mice in the P30 group consumed significantly less energy ELISA. than those in the S30 group (P < 0.001). In mice fed with control diets, there was no significant difference in energy intake between Lymphocyte proliferation the PC and SC groups. These data indicate that the reduced weight gain in mice that were fed with PNO-containing HFDs Overall, the proliferative response of splenocytes to Con A was mainly due to a decrease in adiposity, and might be partly at 0.5 mg/L tended to be higher in mice fed with HFDs compared mediated by a reduction in food intake. with those that were fed with control diets (P = 0.08), and consumption of PNO generally tended to result in higher proliferative responses of splenocytes to Con A at 0.5 mg/L (P = Serum leptin 0.09) (Table 4). The proliferative response of splenocytes to Con Overall, serum leptin levels were higher in mice fed with A at 1.5 mg/L or to LPS at 5, 15, and 30 mg/L was not signifi- HFDs than those fed with control diets (P < 0.001), and PNO cantly affected by the amount or type of fat used in the diets. replacement resulted in lower serum leptin levels (P < 0.001) (Fig. 1). Mice in the P10 group had significantly lower serum Cytokine and PGE2 production leptin levels than those in the S10 group (P =0.04). The serum leptin level showed a significantly positive correlation with Overall, the production of IL-1β by splenocytes, stimulated weight gain (r = 0.88, P < 0.001) and white adipose tissue weight with 10 mg/L of LPS, was significantly higher in mice fed with (r = 0.90, P < 0.001), supporting the contention that PNO PNO (P = 0.04); whereas, the amount of fat did not have a decreased adiposity. significant effect (Table 5). There was no significant influence of the amount or type of fat on the production of IL-2, IFN-γ,

IL-6, or PGE2. 356 Impact of Korean pine nut oil on immune function

Table 4. The proliferative responses of splenocytes from mice fed with control diets or HFDs containing PNO or SBO (× 103 dpm) SBO PNO Effect of fat Effect of fat Control High-fat Control High-fat amount, type, SC S10 S20 S30 PC P10 P20 P30 P value P value (n = 10) (n = 11) (n = 11) (n = 12) (n = 11) (n = 11) (n = 10) (n = 12) Con A, mg/L 0.5 134 ± 22 194 ± 20 191 ± 17 189 ± 20 180 ± 17 220 ± 19 209 ± 21 198 ± 24 0.08 0.09 1.5 158±27 201±28 198±19 188±24 202±16 195±17 152±23 194±26 0.75 0.98 LPS, mg/L 5 70 ± 10 72 ± 8 83 ± 9 74 ± 7 83 ± 8 66 ± 10 71 ± 8 63 ± 11 0.66 0.48 15 74 ± 11 80 ± 8 86 ± 9 78 ± 9 89 ± 10 75 ± 10 78 ± 9 70 ± 12 0.83 0.87 30 68 ± 14 82 ± 12 84 ± 11 73 ± 13 84 ± 12 69 ± 13 76 ± 12 66 ± 14 0.85 0.74 Values are presented as the mean ± SE. A two-way ANOVA was used to determine the effects of fat amount and type. 5 Splenocytes (4 × 10 cells/well) were stimulated with Con A or LPS in 96-well flat bottomed plates for 72 hours at 37℃ in the presence of 5% CO2. Final concentrations of mitogens were 0.5 and 1.5 mg/L for Con A and 5, 15 and 30 mg/L for LPS. Lymphocyte proliferation was measured by assessing [3H] thymidine incorporation during the last 4 hours of incubation. The results are reported as dpm, which are the average dpm of mitogen-stimulated wells minus the average dpm of the wells without mitogens.

Table 5. The production of cytokines and PGE2 by splenocytes from mice fed with control diets or HFDs containing PNO or SBO SBO PNO Effect Effect Control High-fat Control High-fat of fat amount, of fat type, SC S10 S20 S30 PC P10 P20 P30 P value P value (n = 10) (n = 11) (n = 11) (n = 12) (n = 11) (n = 11) (n = 10) (n = 12) Con A, 5 mg/L IL-2 (ng/L) 217 ± 37 292 ± 54 287 ± 59 237 ± 48 189 ± 26 223 ± 33 239 ± 60 199 ± 24 0.47 0.15 IFN-γ (μg/L) 1.9 ± 1.0 1.4 ± 0.6 1.6 ± 0.7 1.2 ± 0.4 1.7 ± 0.6 1.1 ± 0.4 2.9 ± 2.3 1.2 ± 0.4 0.66 0.76 LPS, 10 mg/L IL-6 (μg/L) 1.7 ± 0.2 1.9 ± 0.2 2.2 ± 0.1 2.0 ± 0.1 2.0 ± 0.1 2.1 ± 0.2 2.1 ± 0.2 2.0 ± 0.2 0.36 0.43 IL-1β (ng/L) 38 ± 9a 52 ± 18ab 41 ± 11ab 53 ± 18ab 53 ± 13ab 71 ± 20ab 72 ± 16ab 82 ± 15b 0.55 0.04

PGE2 (ng/L) 683 ± 171 637 ± 81 644 ± 97 475 ± 81 451 ± 73 563 ± 66 864 ± 399 597 ± 159 0.57 0.94 Values are presented as the mean±SE. A two-way ANOVA was used to determine the effects of fat amount and type, followed by an LSD post-hoc test. Means in a row without a common superscript represent significant differences (P < 0.05). Splenocytes (5 × 106 cells/well) were stimulated with Con A (5 mg/L, final concentration) for 48 hours or LPS (10 mg/L, final concentration) for 24 hours in 24-well plates at 37℃ in a 5% CO2 atmosphere. Cell-free supernatants were collected, and the levels of IL-2, IFN-γ, IL-6, IL-1β, and PGE2 were measured by ELISA.

Discussion proliferation observed upon Con A stimulation at 0.5 mg/L in mice fed with PNO was due to less weight gain and lower adipose PNO has been reported to have favorable effects on lipid tissue weight because obese mice, compared with the control metabolism [10,19], blood pressure [5], appetite control [6,7], mice, tended to have higher lymphocyte proliferation upon treat- and eicosanoid production [11]. However, a few studies have ment with Con A at 0.5 mg/L as well. been conducted to determine the effects of PNO on the immune It has been reported that obesity could contribute to impaired function [12]. We focused on whether PNO consumption could immune response [20]. Sato Mito et al. [21] and Moriguchi et influence weight gain, and whether the impact of PNO on al. [22] reported that the proliferative response to T cell mitogens reduced weight gain resulted in an improvement in the immune was significantly lower in obese animal models. However, we function in HFD-induced obese mice. We showed that mice fed did not observe diminished proliferative responses in lymphocytes with PNO-containing HFD gained less body weight and had less from obese mice. Instead, the lymphocyte proliferative response adipose tissue than those fed with SBO-containing HFD. However, to Con A at 0.5 mg/L tended to be higher in obese mice compared dose-dependent effects of PNO on weight gain and white adipose with the lean control mice. Differences in the final body weight tissue weight were not observed among the different doses of and age of animals could be the reasons for discrepancies between PNO tested in this study. results by us and by Sato Mito et al. [21] or Moriguchi et al. PNO consumption tended to increase lymphocyte proliferation [22]. In our study, 5-week-old mice were fed with HFD with when the cells were stimulated with Con A at 0.5 mg/L; however, 45% energy fat for 12 weeks to induce obesity. The average it did not have any significant influence on lymphocyte prolifera- final body weight of obese mice was 16% higher than that of tion when the cells were stimulated with Con A at 1.5 mg/L control mice (36.8 g in obese mice vs. 31.6 g in control mice). or with LPS at 5, 15, or 30 mg/L. Thus, it is inconclusive whether In the study by Sato Mito et al. [21], 4-week-old mice were consumption of PNO will result in improved lymphocyte prolife- fed with HFD with 50% energy fat for 7 months, and obese ration. Additionally, we cannot conclude that higher lymphocyte mice weighed about 2 times more than control mice (45.7 g in Soyoung Park et al. 357 obese mice vs. 23.3 g in control mice) at the end of the possibility that the levels of inflammatory markers measured in experiment. In the study by Moriguchi et al. [22], genetically this study could be affected by α-linolenic acid in SBO diets. obese 8-week-old Zucker rats were obtained and maintained for However, in studies which showed that α-linolenic acid decreased 10 months. Obese rats weighed about 3 times more than control eicosanoid production using a mouse model, the amount of α rats at the end of the experiment (833 g in obese rats vs. 256 g -linolenic acid included was 5.0-7.3 g/100 g diet [29-31]. In this in control rats). Mito et al. [23] reported that proliferation of study, the amount of α-linolenic acid included in SBO diets were splenocytes in response to T cell mitogens in diet-induced obese 0.3 g/100 g diet in the SC group, 0.7 g/100 g diet in the S10 mice was higher, but not statistically significant compared to that group, 1.0 g/100 g diet in the S20 group, and 1.3 g/100 g diet of lean mice. In that study, 4-week-old mice were fed with HFD in the S30 group. Therefore, the amounts in SBO diets do not with 50% energy fat for 13 weeks and the average final body reach the levels that have been reported to have an anti- weight of obese mice was 31% higher than that of control mice inflammatory influence. Except for IL-1β, significant differences (30.8 g in obese mice vs. 23.5 g in control mice), which is were not observed in the inflammatory markers in this study. consistent with our results. In young (24- to 34-year-old) obese In addition, PGE2 levels were also not significantly different human subjects, who did not have metabolic disorders, peripheral between the two oil groups, and dose-dependent changes were blood mononuclear cell proliferation in response to T cell mitogens not observed in mice fed with SBO diets. Thus, anti-inflam- was not significantly different from that of normal weight matory effect of α-linolenic acid as an n-3 PUFA seemed to be subjects [24]. It seems that mild obesity does not significantly minimal with the amount included in SBO diets. suppress the lymphocyte proliferative response, especially in In the current study, we showed that the consumption of HFD, early adulthood. partially substituted with PNO for 12 weeks, led to reduced Ahmed et al. [16] reported that 7% and 10% weight reduction weight gain, which was mainly due to less white adipose tissue after 10% and 30% energy restriction, respectively, enhanced T in the mice. The effect of lower weight gain did not result in cell-mediated immune responses in obese subjects. Tanaka et al. an overall enhancement of the proliferative responses of splenocytes. [17] reported that the altered T cell function observed in obese As the production of IL-1β by splenocytes was higher in mice subjects was corrected after 26% weight reduction. In the current fed with PNO, there is a possibility that PNO has the effect of study, mice fed with HFD with PNO consumed 4% less energy activating the immune response; however, further studies are and weighed 10% less than those fed with HFD containing SBO. needed to determine the mechanisms of the effect of PNO on However, reduced weight gain did not result in an overall IL-1β production, and whether PNO affects the production of improvement in lymphocyte proliferative responses to T cell other inflammatory markers. mitogens. In most studies using animal models, beneficial effects of energy restriction on immune responses was observed when animals were fed 20% to 60% less food [25]. The magnitude References of the reduction in energy intake and weight gain in this study seemed to be insufficient to have a significant influence on 1. Rural Development Administration, National Academy of lymphocyte proliferation. Agricultural Science. Food Composition Table. 8th rev. ed. 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